(a) Field of the Invention
The present invention relates generally to atomic force microscopy (AFM), a cantilever for AFM, and an apparatus and a measuring method of intermolecular interaction between the biomolecules using the same. The present invention regards the usage of dendron coated Bio-AFM tips in measuring the interaction force between biomolecules. The present invention also provides details on Bio-AFM Force Mapping of cell receptors by using surfaces with controlled meso spaces.
(b) Description of the Related Art
In the post-genomic era, quantitative and comprehensive studies on genome for drug discovery, as well as disease diagnosis and prevention, are fast-growing research and development areas. Growth in these sectors has already produced a strong demand for advanced biomolecular recognition probes with high sensitivity and excellent specificity (K. Wang et al., Anal. Chem. 76, 5721 2004).
Out of many biomolecular recognition studies, understanding the mechanical stability (or recognition property) of complementary DNA strands is crucial for a profound understanding of numerous important biological processes, such as DNA transcription, gene expression and regulation, and DNA replication. In this respect, stretching and force-induced melting of DNA have thus been investigated using several techniques, such as optical tweezers, micro-pipette suction, and AFM (H. Clausen-Schaumann, M. Seitz, R. Krautbauer, H. E. Gaub, Curr. Opin. Chem. Biol. 4, 524, 2000; R. Merkel, Physics Reports 346, 343, 2001; G. U. Lee, L. A. Chris, R. J. Colton, Science 266, 771, 1994).
As it is possible to measure specific interactions between individual molecules at small length scales and high sensitivity down to forces of a few piconewtons, AFM is becoming a rapidly developing technique for probing affinity and recognition properties at the molecular level (R. Krautbauer, M. Rief, H. E. Gaub, Nano Lett. 3, 493, 2003). Compared with other sensitive methods for force measurements, AFM has the advantages of high force resolution and high spatial resolution, and is operable under physiological conditions for investigation of specific interactions in biological processes, such as electrostatic interactions (J. Wang, A. J. Bard, Anal. Chem. 73, 2207, 2001), ligand-receptor binding (S. M. Rigby-Singleton et al., J. Chem. Soc., Perkin Trans. 2 1722, 2002), antigen-antibody interactions (F. Schwesinger et al., Proc. Natl. Acad. Sci. U.S.A. 97, 9972, 2000), aptamer-protein interactions (C. Bai et al., Anal. Chem. 75, 2112, 2003), protein folding/unfolding (P. M. Williams et al., Nature 422, 446, 2003; M. S. Z. Kellermayer, S. B. Smith, H. L. Granzier, C. Bustamante, Science 276, 1112, 1997), cell-cell adhesion (M. Benoit, D. Gabriel, G. Gerisch, H. E. Gaub, Nature Cell Biol. 2, 313, 2000) and DNA-DNA hybridization (C. W. Frank, Biophys. J. 76, 2922, 1999).
Although many investigations have been performed on unbinding force measurement between complementary DNA strands (H. Clausen-Schaumann, M. Seitz, R. Krautbauer, H. E. Gaub, Curr. Opin. Chem. Biol. 4, 524, 2000; R. Merkel, Physics Reports 346, 343, 2001; G. U. Lee, L. A. Chris, R. J. Colton, Science 266, 771. 1994; R. Krautbauer, M. Rief, H. E. Gaub, Nano Lett. 3, 493, 2003), the recognition between the DNA strands during the studies at the single molecular level is problematic. The typical immobilization approach suffered from multi-point interaction, and resolving out single molecular interactions has not been an easy task. In order to avoid the unwanted interaction, surface density was reduced by mixing with an inactive surfactant, but the approach resulted in low recognition efficiency leading to less reliable analysis. Therefore, commonly practiced surface chemistry for such immobilization such as oxide-silane and gold-thiol chemistry (T. Hugel, M. Seitz, Macromol. Rapid. Commun. 22, 989, 2001; W. K. Zhang, X. Zhang, Prog. Polym. Sci. 28, 1271, 2003) has yet to be optimized to retrieve invaluable fundamental information on single DNA-DNA interaction during the force measurement with AFM.
Atomic Force Microscopy has traditionally played an important role in understanding the various interaction mechanisms between biomolecules present inside organisms. Through its ability to analyze interaction forces, its importance within the fields of nano and biotechnology is expected to increase into the future as more studies are conducted on the molecular level.
Taking advantage of this technology, many efforts have been made to investigate interactive mechanisms between biomolecules on different surfaces. Unlike liquid, however, observation of biological material on surfaces produce unique problems such as involuntary adsorption and steric hindrance. Among the many measures taken to counter such problems, the most popular one involve measuring monomolecular interaction by applying complex self-assembling film onto surfaces. When observing the singular interaction force between two biomolecules with AFM, two issues come to the forefront. The first problem regards the difference in biomolecular activity on a surface as opposed to within the body. The second issue hinges around the fact that monomolecular interaction cannot be guaranteed in such a setting. Prior research has shown that both problems can be addressed by using self-assembling film technology and meso space manipulation technology (Langmuir 2005, 21, 4257, WO 2006/016787) The technology involves observing intermolecular interactions on the monomolecular level by controlling the spacing and number of biomolecules through limitation of the number of functional groups on which the molecules can be introduced. The most common problem of this method involves the unintended attraction between molecules of the same functional group, resulting in phase separation. In addition, there is no concrete evidence that the biomolecules are evenly spaced between each other.
The importance of biomolecular research using Bio-AFM technology is growing rapidly. Its ability to observe nonconductive material such as biomolecules on the nanometer level in a liquid environment that supports biological activity has made Bio-AFM an important tool in studying the structure and substructures of biological molecules. Additionally, Bio-AFM enables close observation of molecular interaction by its Bio-AFM tip, onto which a biomolecule can be loaded. Important applications include observation of interactions between complementary DNA molecules, mutual interactions between proteins, ligand-receptor interactions, the latter of which holds significance in studying immunological responses to drugs. In researching interaction forces between biomolecules on the monomolecular level, high sensitivity is of primary importance. Monomolecule observation can be accomplished through methods such as Bio-AFM, optical tweezing and magnetic tweezing. Each method comes with its own shortcomings, such as loss of accuracy under the magnetic tweezing method and potential damage that can be incurred upon molecules under the optical tweezing method.
In order to research ligand-receptor mechanisms using Bio-AFM, a ligand needs to be loaded on top of the tip. Generally this is accomplished by utilizing biotin-streptavidin interactions or by use of compound self-assembly films. However, such methods cannot directly control molecular distancing, and can cause ligands to concentrate in certain areas, posing difficulties in observing ligand-receptor interactions with accuracy.